Chemical Profile Analysis and Comparison of Two Versions of the Classic TCM Formula Danggui Buxue Tang by HPLC-DAD-ESI-IT-TOF-MS n

Transcription

1 Molecules 2014, 19, ; doi: /molecules Article OPEN ACCESS molecules ISSN Chemical Profile Analysis and Comparison of Two Versions of the Classic TCM Formula Danggui Buxue Tang by HPLC-DAD-ESI-IT-TOF-MS n Ya-Zhou Zhang 1,2, Feng Xu 3, Tao Yi 1, Jian-Ye Zhang 1, Jun Xu 1, Yi-Na Tang 1, Xi-Chen He 1, Jing Liu 1 and Hu-Biao Chen 1, * School of Chinese Medicine, Hong Kong Baptist University, 7 Baptist University Road, Kowloon, Hong Kong, China Guizhou College of Technology, No.1 Caiguan Road, Guiyang , China State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, 38 Xueyuan Road, Beijing , China * Author to whom correspondence should be addressed; hbchen@hkbu.edu.hk; Tel.: ; Fax: Received: 5 March 2014; in revised form: 10 April 2014 / Accepted: 19 April 2014 / Published: 30 April 2014 Abstract: Danggui Buxue Tang (DBT) is a Traditional Chinese Medicine (TCM) formula primarily used to treat symptoms associated with menopause in women. Usually, DBT is composed of one portion of Radix Angelicae Sinensis (RAS) and five portions of Radix Astragali (RA). Clinically, Radix Hedysari (RH) is sometimes used by TCM physicians to replace RA in DBT. In order to verity whether the chemical constituents of the DBT1 (RA:RAS = 5:1, w/w) and DBT2 (RH:RAS = 5:1, w/w) share similarities the chemical profiles of the two DBTs crude extracts and urine samples were analyzed and compared with the aid of HPLC-DAD-ESI-IT-TOF-MS n, which determines the total ion chromatogram (TIC) and multi-stage mass spectra (MS n ). Then, the DBT1 and DBT2 were identified and compared on the basis of the TIC and the MS n. In the first experiment (with crude extracts), 69 compounds (C1 C69) were identified from the DBT1; 46 compounds (c1 c46) were identified from the DBT2. In the second experiment(with urine samples), 44 compounds (M1 M44) were identified from the urine samples of rats that had been administered DBT1, and 34 compounds (m1 m34) were identified from the urine samples of rats that had been administered DBT2. Identification and comparison of the chemical compositions were carried out between the DBT1 and DBT2 of the crude extracts and urine samples respectively. Our results showed that the two crude extracts of the DBTs have quite

2 Molecules 2014, different chemical profiles. The reasons for their differences were that the special astragalosides in DBT1 and the isoflavonoid glycosides formed the malonic acid esters undergo single esterification and acetyl esters undergo acetylation in DBT1. In contrast, the urine from DBT1-treated rats strongly resembled that of DBT2-treated rats. These metabolites originate mainly from formononetin, calycosin and their related glycosides, and they were formed mainly by the metabolic process of reduction, deglycosylation, demethylation, hydrogenation and sulfation. The HPLC-DAD-ESI-IT-TOF-MS n method was successfully applied for the rapid chemical profiles evaluation of two DBTs and their related urine samples. Keywords: Radix Hedysari; Radix Astragali; Danggui Buxue Tang; HPLC-DAD-ESI-IT- TOF-MS n ; isoflavonoid; astragaloside 1. Introduction Danggui Buxue Tang (DBT) is a Traditional Chinese Medicine (TCM) formula primarily used to treat symptoms associated with menopause in women. It is believed to invigorate Qi (vital energy) and nourish the Blood (body circulation) [1]. Nowadays, it is commonly used in China as an efficacious medicinal prescription and a healthy food supplement. Pharmacological studies have found that DBT promotes hematopoietic function [2,3], regulates blood lipid and anti-inflammatory activities in diabetic atherosclerosis [4,5], anti-fibrosis effects [6], prevents osteoporosis [7,8], and increases anti-oxidation activity as well as immune response [9]. According to its original formula, DBT comprises Radix Astragali (RA) and Radix Angelicae Sinensis (RAS) (5:1, w/w). More recently, Radix Hedysari (RH) has been used to replace RA. Thus, in current clinical applications, DBT is prescribed in two forms: RA:RAS (5:1) (called DBT1), and RH:RAS (5:1) (called DBT2) [1,10]. The plants RA and RH belong to the same botanical family but different genus, and have long been widely used as the same crude herb in DBT [1]. This is always a question of whether RH can replace RA in the DBT decoction. Chemically, RA-containing DBT showed higher amounts of calycosin-7-oβ-d-glucoside, ferulic acid, ononin, calycosin, astragaloside IV, astragaloside III, and Z-ligustilide. Only formononetin was higher in RH-containing DBT. In parallel, the estrogenic, osteogenic and erythropoetic effects of RA-containing DBT1 showed better activities than that of RA-containing DBT2 [1]. So far, the chemical differences between DBT1 and DBT2 has not been investigated. Therefore, we designed a systematic comparison of the chemical ingredients of DBT1 and DBT2. Two experiments were designed, including thorough elucidation of the chemical profiles of DBT1 and DBT2 crude extracts and illumination of the metabolites of DBT1 and DBT2 after being administrated to rats. The chemical profiles of the two DBTs were compared by determining the total ion chromatogram(tic) and the multistage mass spectra (MS n ) from HPLC-DAD-ESI-IT-TOF-MS n. Subsequently, DBT1 and DBT2 were identified and compared on the basis of the TIC and the MS n [11]. The results will be provide a solid evidence to understand the chemical profiles of the two different versions of DBT.

3 Molecules 2014, Results and Discussion 2.1. Optimization the Conditions of HPLC and Mass Spectrometry In order to obtain desirable HPLC and mass spectrometry chromatograms, the procedures for preparation of the urine samples and crude extracted samples of the two DBTs were optimized in terms of the extraction solvents and extraction times. Methanol and acetonitrile were initially selected as the extraction solvents, but methanol is less poisonous and produced almost the same chromatograms as acetonitrile, so it was applied as the final extraction solvent. For comparison, different columns (Phenomenex RP C 18, Agilent RP C 18 ) were tested for sample separation, and Phenomenex RP C 18 gave the best chromatographic resolution. The column was eluted with a gradient mobile phase that consisted of water-formic acid (100:0.1, v/v) (A), acetonitrile (B) and at a flow rate of ml/min, in addition, 0.1% (v/v) formic acid was added to improve the mass spectrometry ionization efficiency and enable symmetric peak shapes [12]. Both the positive ion (PI) and negative ion(ni) modes were tested for the experiment. Since MS and MS n fragmentations gave more information about the isoflavones in PI mode but about saponins in NI mode, the analysis was simultaneously conducted in both PI and NI mode The Identification and Analysis of 19 Reference Compounds Nineteen reference compounds which might represent the major structural types of the DBTs were analyzed. The characteristic fragment ions of 19 references are very useful for determining the structural skeleton and the substitution patterns of those related compounds in two DBTs. Their high resolution mass spectra (HRMS) data are summarized (see Table 1 and Supplementary: pages S2 S8). The base peak chromatograms (BPCs) detected in NI and PI mode were recorded (see Figure 1a). Furthermore, the structure of 19 reference compounds were shown (Figure 1b) [11,13]. According to our preliminary research, there three main types of isoflavonoids are found in Radix Astragali which were named as a: isoflavone (T1, T2, T5, T6, and T8), b: isoflavan (T4, T7, and T10) and c: pterocarpan (T3, and T9) [11,13 15]. To facilitate the structural identification of the isoflavonoids in the DBTs, the fragmentation behaviors of the three types of isoflavonoids were analyzed, which might represent the major structural types. We first studied the MS n fragmentation behaviors in PI and NI mode, and found that the fragmentation behaviors in PI mode could give more information about the structure than in NI modes. Then we elucidated the structure of the three types of isoflavonoids mostly from the PI mass spectra. Isoflavone had the characteristic fragment ions 5 B + -2H, 0,3 B + -2H, 5 A + -2H, 1,3 A + -2H, 3,4 A + -2H, etc., isoflavan had the characteristic fragment ions 5 B + -2H, 5 A + -2H, 1,3 A + -2H, etc., and pterocarpan had the characteristic fragment ions 6,7 B + -2H, 1,4 B + -2H, 3,4 A + -2H, 5,6 A + -2H, etc., based on MS 2 and MS 3 spectra by HPLC-DAD-ESI-IT-TOF-MS n (see Figure 2). For example, we identified the characteristic malonate-glucose-, acetyl-glucose- and glucose-binding ingredients with a neutral loss of 248 Da, 204 Da, and 162 Da, otherwise, glucuronide metabolites with a neutral loss of ( 176 Da) and sulfated metabolites with a neutral loss of ( 80 Da) from the molecular ion peaka in the MS 2 spectra [13,16,17].

4 Molecules 2014, NO. T R (min) Table 1. The fragment ions of 19 kinds of reference compounds by HPLC-DAD-ESI-IT-TOF-MS n. [M+H] + [M H] Predicted Formula Fragment Ions Da Error (ppm) The Name of the Reference Compounds 1(T1) C 22 H 22 O ,285,270, Calycosin-7-O-β-D-glucopyranoside 2(AW) C 10 H 10 O 4 193,178, Ferulic acid 3(T2) C 22 H 22 O 9 431,269,253,237,213,197,163,134, Ononin 4(T3) [M+Na] + C 23 H 26 O ,463,323, Astrapterocarpan-7-O-β-D-glucopyranoside 5(T4) [M+Na] + C 23 H 28 O ,303, Astraisoflavan-7-O-β-D-glucopyranoside 6(T5) C 16 H 12 O 5 285,270,253,225,197, Calycosin 7(T6) C 24 H 24 O , ''-O-acetyl-ononin 8(T7) [M+Na] + C 25 H 30 O ,507, ''-O-acetyl-astraisoflavan-7-O-β-Dglucopyranoside 9(T8) C 16 H 12 O 4 269,270, Formononetin 10(T9) C 17 H 16 O 5 301,271,251,167,151, (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan; Astrapterocarpan 11(T10) C 17 H 18 O 5 303,181,167,149, (3R)-7,2'-dihydroxy-3',4'-dimethoxyisoflavan; Astraisoflavan 12(G Ⅴ ) [M+HCOO] C 47 H 78 O ,783, Astragaloside Ⅴ 13(G Ⅳ ) [M+HCOO] C 41 H 68 O ,783,621,489, Astragaloside Ⅳ 14(G Ⅲ ) [M+HCOO] C 41 H 68 O ,783,651, Astragaloside Ⅲ 15(G Ⅱ ) [M+HCOO] C 43 H 70 O ,765, Astragaloside Ⅱ 16(G Ⅰ ) [M+HCOO] C 45 H 72 O ,867, Astragaloside Ⅰ 17(HHQC) [M+Na] + C 30 H 50 O 5 513,515,405, Cycloastragenol(HHQC) 18(ZL) C 12 H 14 O 2 191,173, Z-ligustilide 19(EL) C 12 H 14 O 2 191, E-ligustilide

5 Molecules 2014, Figure 1. (a) LC-MS chromatogram of 19 reference compounds in PI (1BPC) and NI (4BPC) mode. (b) The chemical structures of 19 reference compounds. (a) (b)

6 Molecules 2014, Figure 2. The bond cleavage pathways of the three types of isoflavonoids in RA Profiling and Identifying Chemical Compounds of the Two Crude Extracts (DBT1, and DBT2) by HPLC-DAD-ESI-IT-TOF-MS n Identification of the Chemical Profiles of DBT1 by HPLC-DAD-ESI-IT-TOF-MS n The HRMS data of these identified compounds are summarized (see Table 2 and Supplementary: pages S9 S35). The BPCs detected in PI (1BPC) and NI (4BPC) mode were also recorded (Figure 3a) [17 19]. By comparing the fragment ions and retention times and based on the high resolution mass spectra software predicted formulas with the reference compounds from the MS and MS n, the compounds C23, C33, C36, C37, C38, C44, C46, C56, C58, C59, C49, C53, C60, C64, and C68 were identified as the reference compounds [11,13]. C27 has a RT at min, [M+HCOO] at m/z in MS (predicted formula: C 29 H 38 O 15 : ppm error: 5.66), and characteristic fragment ions at m/z ( 46 Da) [M-H], m/z [M-162-H], and m/z [M H] in MS 2. The neutral loss is mass 46 Da (CH 2 O 2 ; identified as HCOOH), 162 Da*2 (C 6 H 10 O 5 ; identified as glucopyranoside), and the fragment ion m/z predicted as C 17 H 18 O 5. Then C27 was identified as astraisoflavan-di-7-o-β-d-glucoside or its isomer. The [M+H] + or [M-H] of C24, C29, C30, and C41 shows the same neutral loss of 162 Da (C 6 H 10 O 5 ; glucoside) in them MS 2, so both of them were identified as the glycosides [17 19]. The characteristic fragment ions of C31, C35, C43, C42, and C51 have a neutral loss of 248 Da (C 9 H 12 O 8; identified as the 6''-O-malonate-glucoside) in them MS 2, so both of them were identified as glycosides of 6''-Omalonate-glucoside [13].The HRM software predicted [M+H] + or [M-H] of C32, C40, C34, C47, and C18, whose formulas have the same characteristic fragment loss ( 204 Da; C 8 H 12 O 6 ) which was identified as 6''-O-acetylglucosides. In addition, C32, C40, C34, C47, and C18 were identified as glycosides of 6''-O-acetylglucoside [16]. For the predicted formulas of [M+H] + or [M-H], we tentatively identified C39, C50, C62, C30, and C41 as the isoflavonoid-related constituents by referring to the literature [18,19], and C45, C55, C48, C52, C54, C61, and C65 were tentatively identified as being related to saponins [13,17 19]. Using the [M+H] +, [M-H] or [M+Na] + data of C7, C8, C10, C12, C13, C14, C21, C25, C26, and C28, we predicted their formulas, which indicates that they are the ingredients of the samples. However, at this point, their exact structures could not be identified.

7 Molecules 2014, Table 2. The identified proposed compounds of the crude extract samples from Danggui Buxue Tang 1 and Danggui Buxue Tang 2 by HPLC-DAD-ESI-IT-TOF-MS n. NO. T R (min) [M+H] + [M H] Predicted Formula Fragment Ions Da Error ppm Identification DBT1 DBT C 6 H 14 N 4 O Arginine C1 c C 6 H 12 O Gluconic acid C2 c C 12 H 22 O D(+)sucrose C3 c C 6 H 8 O 7 191, Citric acid C4 c C 11 H 11 N 3 O C5 c C 11 H 11 N 3 O C6 c C 18 H 26 O , C [M+Na] + C 17 H 24 O c C 18 H 18 O 2 267, Magnolol -- c C 18 H 26 O ,351,301, C C 9 H 11 NO L-phenylalanine C9 c C 18 H 24 O , C C 13 H 21 NO C11 c C 19 H 26 O , c [M+Na] + C 26 H 22 O 8 485, C C 8 H 14 O C C 20 H 28 O C C 21 H 22 O , c C 21 H 22 O ,285, c C 17 H 22 O ,285, C15 c C 23 H 32 O C16 c C 18 H 26 O ,269, C17 c C 24 H 24 O ,443, ''-O-acetyl-pratensein-7-O-β-D-glucoside C C 11 H 12 O C19 --

8 Molecules 2014, NO. T R (min) [M+H] + [M H] Predicted Formula Table 2. Cont. Fragment Ions Da Error ppm Identification DBT1 DBT C 21 H 30 O 3 331, C C 18 H 23 N 5 O C C 13 H 24 N 2 O 5 289,272, C22 c C 30 H 24 O 6 479, C [M+HCOO] C 29 H 38 O ,625,463, Astraisoflavan-di-7-O-β-D-glucoside C C 23 H 36 N 2 O ,417, C C 22 H 22 O , Glycetein-4'-O-β-D-glucoside C C 22 H 22 O , Kaempferide-7-O-β-D-glucoside C C 25 H 24 O , ''-O-malonate-calycosin-7-O-β-D-glucoside C C 24 H 24 O , ''-O-acetyl-calycosin-7-O-β-D-glucoside C C 22 H 22 O 9 431,269,237, Ononin C33 c C 24 H 26 O ,285,271, ''-O-acetyl-isosakuranetin-7-O-β-D-glucoside C C 25 H 24 O , ''-O-malonate-kaempferide-7-O-β-D-glucoside C C 23 H 26 O (+Na + ),463, Astrapterocarpan-7-O-β-D-glucopyranoside C C 23 H 28 O ,301, Astraisoflavan-7-O-β-D-glucopyranoside C C 15 H 12 O 4 255, Isoliquiritigenin -- c C 16 H 12 O 5 285,270,225, Calycosin C38 c C 16 H 14 O 4 269,253, Isomer of alpinetin -- c C 15 H 12 O 4 255, Liquiritigenin -- c C 17 H 14 O methoxy-maackiain or the isomer C C 24 H 24 O , The isomer of 6''-O-acetyl-ononin C C 22 H 22 O , Glycetein-7-O-β-D-glucoside C C 26 H 28 O , ''-O-malonate-astrapterocarpan-glucoside C C 25 H 24 O , ''-O-malonate-ononin C C 25 H 30 O , ''-O-acetyl-astraisoflavan-7-O-β-D-glucoside C44 --

9 Molecules 2014, NO. T R (min) [M+H] + [M H] Predicted Formula Table 2. Cont. Fragment Ions Da Error ppm Identification DBT1 DBT C 48 H 78 O ,541, Soyasaponin Ba C45 c C 24 H 24 O , ''-O-acetyl-ononin C C 47 H 76 O Akebia saponin D -- c C 24 H 24 O , ''-O-acetyl-kaempferide-7-O-β-D-glucoside C C 41 H 70 O Cyclocanthoside E C C 48 H 80 O Astragaloside V C C 16 H 14 O 5 285,194, Isomer of isosakuranetin -- c C 15 H 10 O 5 269, Genistein -- c C 17 H 16 O 6 315, Astragaluquinone or isomer C C 25 H 24 O , ''-O-malonate-glycetein-7-O-β-D-glucoside C C 45 H 72 O Isoastragaloside I C C 16 H 12 O 5 283,268, Glycetein -- c [M+HCOO] C 41 H 68 O Astragaloside IV C C 43 H 70 O , Isoastragaloside II C C 18 H 34 O c C 48 H 76 O c C 15 H 12 O Dihydro-kaempferol -- c C 15 H 12 O 4 256, Isomer of Liquiritigenin -- c C 16 H 12 O 4 269,254,237, Formononetin C56 c C 48 H 78 O ,525, Soyasaponin Bb C55 c C 17 H 14 O 5 299,284,166 Pterocarpin -- c C 18 H 34 O C C 16 H 12 O 5 283,255, The isomer of glycetein -- c C 17 H 16 O Astraoptercarpan C58 --

10 Molecules 2014, NO. T R (min) [M+H] + [M H] Predicted Formula Table 2. Cont. Fragment ions Da Error ppm Identification DBT1 DBT C 17 H 18 O Astraisoflavan C [M+HCOO] C 43 H 70 O ,825, Astragaloside II C [M+HCOO] C 45 H 70 O , C C 16 H 12 O Kaempferide or isomer -- c C 47 H 74 O Acetylastragaloside I -- c [M+HCOO] C 46 H 74 O Castaraleside H -- c C 15 H 10 O 6 285, Kaempferol or isomer C C 48 H 76 O c C 16 H 12 O 5 285,253, Isomer of calycosin -- c [M+Na] + C 18 H 32 O C63 c [M+HCOO] C 45 H 72 O Astragaloside I C C 12 H 16 O Senkyunolide A -- c [M+HCOO] C 47 H 72 O , C [M+Na] + C 28 H 46 O C C 12 H 14 O n-butyl-phthalide -- c [M+Na] + C 12 H 14 O 2 403, Z-ligustilide C67 c [2M+Na] + C 12 H 14 O 2 403,381, E-ligustilide C68 c C 15 H 8 O 6 283,203, C69 c46

11 Molecules 2014, Figure 3. (a) The BPC in NI and PI mode of the crude extracts of Danggui Buxue Tang 1 (RA:RAS = 5:1). (b) The BPC in NI and PI mode of the crude extracts of Danggui Buxue Tang2 (RH:RAS = 5:1). (c) The BPC in NI and PI mode of the urine samples of rats that had been administrated the Danggui Buxue Tang 1 (RA:RAS = 5:1) and (d) The BPC in NI and PI mode of the urine samples of rats had been administrated the Danggui Buxue Tang 2 (RH:RAS = 5:1). (x10,000,000) 1:BPC (1.00) 4.0 4:BPC (1.00) 3.0 (a) (x10,000,000) 1:BPC (1.00) 3.0 4:BPC (1.00) C (x10,000,000) 4.0 1:BPC (1.00) 4:BPC (1.00) c5 c6 C9 C15 C16 C17 c14 C22 c16 M2 M3 M4 M5 M6 C33 c18 M21 M23 M22 M24 M M9 C38 C45 c20 c23 M14 M17 C56 c28 C57 c32 c35 C63 c41 (b) (c) (x10,000,000) 1:BPC (1.00) 4:BPC (1.00) 4: (66.21) 5.0 4: (22.80) m7 (d) 2.5 m2 m4 m3 m m9 m13 m15 m18 m20 m19 m21 m22 m Identification of the Chemical Profiles of DBT2 by HPLC-DAD-ESI-IT-TOF-MS n The HRMS data of these identified compounds are summarized (see Table 2 and Supplementary: pages S36 S51). The BPCs detected in NI and PI modes were recorded (see Figure 3b). Using their fragment ions and retention times in MS data, c18, c20, c32, c44, and c45 were identified as the reference compounds [11,13]. Based the MS data, c12 shows RT at min, [M-H] at m/z in MS (predicted the formula: C 21 H 22 O 10 : ppm error: 2.54), and characteristic fragment ions at m/z ( 148 Da; C 5 H 8 O 5 ; identified as the ribonic acid) and predicted as C 16 H 14 O. Compound c12 was identified as the isomer of isosakuranetin-ribonic acid. Moreover, c13 shows a characteristic neutral loss at 148 Da (C 5 H 8 O 5 ) with the same as c12 [16]. By the formulas predicted of [M-H] or [M+H] + and referring to literature [13,17 19], c23, c24, c29, c35, c38, and c39 were tentatively identified as saponin-related constituents, and c7, c8, c11, c19, c21, c22, c25, c26, c27, c31, c33, c34, c36, c40, and c43 were tentatively identified (see in Table 2). By using the HRMS data (RT, Predicted the formulas and characteristic fragment ions) compared with the BDT1 crude extract samples, C1, c1; C2, c2; C3, c3; C4, c4; and C9, c9 were identified as the same constituents [17 19].

12 Molecules 2014, The groups of C5, c5; C6, c6; C11, c10; C15, c14; C16, c16; C17, c15; C22, c17; C57, c28; C63, c41; and C69, c46 between DBT1 and DBT2 were tentatively identified as the same compounds with uncertain structures. From the analysis based on the comparison of TIC and MS n : 69 compounds (C1 C69) were identified from the crude extracts of DBT1, 46 compounds (c1 c46) were identified from the crude extracts of DBT2. The isoflavonoids glycosides had experienced acetylation (seven compounds, C18, C32, C34, C40, C44, C46, and C47), formed the malonate acid esters (five compounds, C31, C35, C42, C43, and C51) and with special astragalosides (six compounds, C49, C52, C53, C54, C60, and C64) in DBT1. Thus, the number of identified components in DBT1 was significantly more than in DBT2 (the chemical structural diversity of isoflavonoids which were detected in DBT1 more than in DBT2 are shown in Figure 4). Among these, the 24 common chemical constituents accounted for approximately 27% to the total 91 identified compounds. However, and the proportion of the total isoflavonoids and saponins to the total identified ingredients accounted for nearly 62% (see Table 3). Figure 4. The chemical structures of the main proposed different isoflavonoids in Danggui Buxue Tang 1 more than Danggui Buxue Tang 2.

13 Molecules 2014, Table 3. The number comparison of the identified compounds between Danggui Buxue Tang 1 and Danggui Buxue Tang 2. N. ID. S-ID. T.ID. S% H + S S% DBT1 69 DBT = DBT1-U 44 DBT2-U = ID. Total identified proposed compounds number; S-ID. Identified the common proposed compounds number between two Danggui Buxue Tangs; T.ID. Identified the unfamiliar proposed compounds number between two Danggui Buxue Tangs; H + S: The total isoflavones and the total saponins number; S% The ratio about the selective compounds in the total identified proposed compounds number Profiling and Identifying Chemical Profiles of the Urine Samples after Administration of the DBT1 and DBT2 Performed by HPLC-DAD-ESI-IT-TOF-MS n In the study of the existing literature, ingredients such as isoflavones, saponins in the two DBTs had no obvious differences in chemical profiles between the serum and bile samples collected from enterohepatic circulation. In addition, they had a lower concentration in serum samples, even when giving at dosages of g/kg (w/w) several times to rats within 24 h [13,17,20]. Thus, this approach is not conducive to tracing these minor components. This study chose the normal usage of 10 g/kg (w/w) by comparing the urine samples of rats that were administrated two different DBTs, so as to improve the detection through enrichment of the treatments Identification of the Chemical Profiles of Urine Sample after Administration of the DBT1 by HPLC-DAD-ESI-IT-TOF-MS n The HRMS data of these identified metabolites are summarized (see Table 4 and Supplementary: pages S52 S66). The BPCs detected in NI mode were recorded (see Figure 3c). In addition, the main proposed structures of these metabolites identified from the urine samples of rats that had been administrated DBT1 were showed (see Figure 5). Using MS data with the reference compounds, M15 was identified as calycosin, and M23 was identified as formononetin [11,13]. With the predictions of [M-H] or [M+HCOO], and the characteristic fragment ions, M9, M7, M19, M8, M10, M11, M13, M14, M22, M16, M18, M20, M21, M24, M32, and M36 were tentatively identified as the metabolites of isoflavonoids [13,17 19,21]. M25 shows RT at min, [M+H] + at m/z in MS (predicted formula: C 15 H 10 O 7 S: ppm error: 5.67), and characteristic fragment ions at m/z ( 80 Da; SO 3 ; identified as the sulfonyl hydroxide) and predicted as C 15 H 10 O 4. Then M25 was identified as daidzein after sulfation. M27, M26, M34, M28, M29, M30, M31, M33, M35, M37, M38, M43, and M44 have the same neutral loss of 80 Da, which was identified as the sulfonyl hydroxide (SO 3 ), so they were identified as the sulfated products [13,16]. When predicting their formulas, M39, M41, and M42 were identified as related metabolites of saponins [13,17].

14 Molecules 2014, Table 4. The identified proposed metabolites from the urine samples of rats that had been administrated Danggui Buxue Tang1 and Danggui Buxue Tang 2. NO. T R (min) [M+H] + [M-H] Predicted Formula Fragment Ions Error ppm Identification DBT1 DBT C 10 H 8 O M1 m C 12 H 12 N 2 O 3 463, M2 m C 12 H 2 N 4 O 2 233, M3 m C 16 H 22 N 2 O 6 337, M4 m C 13 H 17 NO m C 13 H 8 O 7 275, M5 m C 13 H 6 O 7 273, M6 m C 15 H 10 O Hydroxydaidzein M [M+HCOO] C 22 H 22 O 9 475, Isomer of ononin -- m C 16 H 16 O 6 303, Hydroxylcalycosin, direduction(c 2 =C 3 ; C 4 =O) M C 15 H 10 O Daidzein M9 m C 23 H 26 O , Astraisoflavan, glucuronidation M C 15 H 12 O 4 255, Daidzein, reduction(c 2 =C 3 ) M11 m C 18 H 24 O Hydroligustilide, glucuronidation M C 15 H 12 O Daidzein, reduction(c 4 =O) -- m C 16 H 14 O 5 285,269, Calycosin, reduction(c 2 =C 3 ) M13 m C 15 H 14 O Daidzein, direduction(c 2 =C 3 ; C 4 =O) M C 16 H 12 O 5 283, Calycosin M15 m C 16 H 14 O 5 285, Calycosin, reduction(c 4 =O) M C 15 H 14 O 5 273,240, Hydroxydaidzein, direduction (C 2 =C 3 ; C 4 =O) -- m C 12 H 2 N 4 O M17 m [M+HCOO] C 16 H 14 O Dihydroxycalycosin, reduction(c 2 =C 3 ) M18 --

15 Molecules 2014, NO. T R (min) [M+H] + [M-H] Predicted Formula Fragment Ions Table 4. Cont. Error ppm Identification DBT1 DBT C 16 H 12 O 5 283,268, Isomer of calycosin m C 15 H 10 O Hydroxydaidzein M C 16 H 12 O 5 283,268, Isomer of calycosin -- m C 16 H 12 O 6 299, Hydroxycalycosin, or isomer M C 16 H 12 O 6 299, Hydroxycalycosin M C 15 H 14 O Isoliquiritigenin, reduction(c=c) M20 m C 16 H 12 O 4 269,253, Formononetin M23 m C 16 H 14 O 4 269,254, Formononetin, reduction(c 2 =C 3 ) M24 m C 15 H 10 O 7 S 335, Daidzein, sulfation M25 m C 16 H 12 O 6 299, Hydroxycalycosin -- m C 16 H 12 O 8 S 363, Calycosin, sulfation M C 15 H 10 O 7 S 333,253, Daidzein, sulfation M C 16 H 14 O 8 S 365, Calycosin, reduction(c 2 =C 3 ), sulfation M C 15 H 10 O 7 S 333,253, Daidzein, sulfation -- m C 15 H 10 O 7 S 333, Isomer of daidzein, sulfation -- m C 16 H 14 O 8 S 365, Calycosin, reduction(c 2 =C 3 ), sulfation M C 15 H 10 O 8 S 349,269, Hydroxydaidzein, sulfation M28 m C 15 H 12 O 8 S 351,271, Hydroxydaidzein, reduction(c 2 =C 3 ), sulfation M C 15 H 14 O 7 S 337, Daidzein,direduction(C 2 =C 3 ; C 4 =O), sulfation -- m C 16 H 12 O 5 283, Isomer of calycosin M C 16 H 16 O 8 S 367,272, Calycosin, direduction(c 2 =C 3 ; C 4 =O), sulfation M C 16 H 12 O 8 S 363, Calycosin, sulfation M34 m C 15 H 12 O 7 S 335,255, Daidzein, reduction(c 2 =C 3 ), sulfation M35 --

16 Molecules 2014, NO. T R (min) [M+H] + [M-H] Predicted Formula Fragment Ions Table 4. Cont. Error ppm Identification DBT1 DBT C 18 H 16 O M C 15 H 12 O 8 S 351, Hydroxydaidzein, reduction(c 2 =C 3 ), sufaltion -- m C 15 H 14 O 6 S 321, Equol, sulfation M37 m C 15 H 14 O 7 S 337,257, Daidzein, direduction(c 2 =C 3 ; C 4 =O), sulfation M [M+Na] + C 36 H 52 O Related to astragaloside -- m C 35 H 64 O Related to astragaloside M C 35 H 58 O Related to astragaloside M C 33 H 54 O Related to astragaloside M41 m C 33 H 50 O Related to astragaloside -- m C 29 H 46 O Related to astragaloside M C 15 H 14 O 8 S 353, Hydroxydaidzein,direduction(C 2 =C 3 ;C 4 =O) sulfation M C 16 H 14 O 10 S 397, Dihydroxycalycosin, reduction(c 2 =C 3 ), sulfation M C 16 H 12 O 8 S 363, Calycosin, sulfation -- m C 16 H 12 O 7 S 347, Formononetin, sulfation -- m34

17 Molecules 2014, Figure 5. The main proposed metabolites identified from the urine samples of rats that had been administrated Danggui Buxue Tang Identification of the Chemical Profiles of Urine Sample after Administration of the DBT2 by HPLC-DAD-ESI-IT-TOF-MS n The HRMS data of these identified metabolites are summarized (see Table 4 and Supplementary: pages S67 S77). The BPCs detected in NI model were recorded (see in Figure 3d). The main proposed structures of metabolites identified from the urine samples of rats that had been administrated DBT2 were showed (see Figure 6). The MS data of m13, and m19 show that they are the reference compounds. In addition, m13 was identified as calycosin, and m19 was identified as formononetin [11,13]. Moreover, m8 has a RT at min, [M+HCOO] at m/z in MS (predicted formula: C 22 H 22 O 9 : ppm error: 0.42), and characteristic fragment ions at m/z that are predicted as C 16 H 12 O 4, the neutral loss is 46 Da (HCOOH) Da (C 6 H 10 O 5 ; identified as glucoside). Thus, m8 was identified as the isomer of ononin [13]. In their MS and MS 2 Data, m21, m23, m22, m24, m25, m26, m33, m27, m28, m29, and m34 have the same neutral loss of 80 Da, which was predicted as the sulfonyl hydroxide (SO 3 ), so they were identified as the sulfated products [13,16].

18 Molecules 2014, Figure 6. The main proposed metabolites identified from the urine samples of rats that had been administrated Danggui Buxue Tang 2. By the MS data of [M-H] or [M+HCOO] and the characteristic fragment ions, m9, m10, m11, m12, m14, m16, m17, m18, and m20 were identified as the metabolites of isoflavonoids [13 15,17,18,21]. Using their predictions of [M+Na] + or [M+H] +, m30, m31, and m32 were tentatively identified as saponin-related metabolites [13,17]. Between DBT1 and DBT2, the groups M1, m1; M2, m2; M3, m3; M4, m4; M5, m6; M6, m7; and M15, m17 were identified as the same compounds, respectively. In this part of the experiment, decoctions of DBT1 and DBT2 were administered to rats, and an analysis was conducted on the rats urine for metabolites: 44 compounds (M1 M44) were identified from the urine samples after DBT1 was administrated, 34 compounds (m1 m34) were identified from the urine samples after DBT2 was administrated. The number of the chemical components in the urine samples from DBT1 was administrated to rats are slightly more than these of DBT2. The proportion of the 19 common constituents to the total 59 identified compounds accounted for approximately 33%. The proportion of 48 total isoflavonoids and saponins to the total identified compounds, however, reached approximately 82% (See Table 3). The phase Ⅱ metabolites from the urine samples of DBT1 and DBT2 were dominantly sulfated products, with rare or no glucuronide metabolites. This result still requires further research as the literature reports the chief presence of mainly glucuronide metabolites [13 16]. Metabolites that originated from RAS are relatively rare or not detected. This is likely due to the low proportion of RAS in DBT and even the low dosage that was given to rats in this study, those chemical constituents of RAS being easy to volatilize, or a loss when sampling was conducted by concentrated processes [22,23]. At this normal dosage of DBT and due to poor absorption, the content levels of astragalosides are

19 Molecules 2014, much lower. In addition, saponins and their metabolites that originated from astragalosides are rarely detected [24 27]. The HPLC-DAD-ESI-IT-TOF-MS n method adopted in our research was confirmed to be a powerful method to evaluate the chemical profiles of the crude extracts and the related urine samples. As we know, the chemical composition found in a Chinese herbal decoction is rather complicated. In this study, the chemical profile analysis of DBT1 and DBT2 was conducted, which provided a comprehensive understanding of that those isoflavonoids that play an important role in the main common chemical basis when they are used in clinical practice. Some identified metabolites are known to have many bioactivities, such as calycosin, formononetin, daidzein and equol (which are well-known phytoestrogens), and most of them displayed many beneficial effects to humans [28 30]. Through the comparison of chemical profiles of two DBTs at our used normal dosage, the similarity of urine samples is higher than that of the crude extract samples. This leads us to believe that the main chemical basis of the chemical constituents is almost the same. Whether the DBT2 of RH:RAS can replace the DBT1 of RA:RAS, we need to further investigate different ratios of RH:RAS with the RA: RAS (5:1) when using equivalent pharmacological research. 3. Experimental 3.1. Materials and Reagents Radix Astragali was collected from Shanxi Province (Voucher No , Specimen No. 1167), Radix Hedysari was collected from Neimeng Province (Voucher No , Specimen No. 1168) and Radix Angelica sinensis was collected from Gansu Province (Voucher No , Specimen No. 1169). All of those medicinal materials were purchased from Guangzhou Zixing Herbal Company in Guangzhou at June in 2013 by Liu Jing and they are identified by Prof. Chen Hu-Biao. The following reference compounds: (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan (astrapterocarpan), ononin, formononetin, 6''-O-acetyl-ononin, calycosin, ferulic acid, (3R)-7,2'-dihydroxy-3',4'-dimethoxyisoflavan (astraisoflavan), calycosin-7-o-β-d-glucoside, astrapterocarpan-7-o-β-d-glucoside, Z-ligustilide, E-ligustilide, astragaloside I, astragaloside II, astragaloside III, astragaloside IV, astragaloside V, astraisoflavan-7-o-β-d-glucoside and 6''-O-acetyl-astraisoflavan-7-O-β-D-glucoside were prepared and identified in our preliminary work [10 14]. Acetonitrile (Merck Co., Darmstadt, Germany) and formic acid (Mreda Technology Inc., Beijing, China) were of HPLC grade. Ultra-pure water was prepared by a Milli-Q water purification system (Millipore, Billerica, MA, USA) Sample Preparation In clinical use, DBT is typically boiled with water twice, then the two decoctions are combined and applied [1]. Therefore, in our study, DBT1, consisting of RA 100 g and RAS 20 g was boiled in 1,000 ml of water (w:v) for 45 min, and then the decoction was filtered. The residue was again boiled in 700 ml of water (w:v) for 30 min. The two decoctions were evaporated to dryness under reduced pressure at 50 C to 100 ml volume. Samples of DBT2, consisting of RH 100 g and RAS 20 g, were prepared in the same way.

20 Molecules 2014, Animals and Administration 12 male Sprague-Dawley (SD) rats ( g) were provided by the Experimental Animal Center of Peking University Health Science Center (Beijing, China) and divided into two groups. They were housed in metabolic cages (Type: DXL-DL, Suzhou Fengshi Laboratory Animal Equipment Co. Ltd., Suzhou, China), and kept in an environmentally controlled breeding room for one-week acclimation. Throughout the experiments, rats had unrestricted access to laboratory chow and water. The DBT1 and DBT2 were administrated by oral at a dose of raw medicinal material 10 g/kg body weight once a day (at 17:30 pm) respectively. Totally for 2 days. All procedures used in the animal experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the US National Institute of Health. The experiments were reviewed by the Biomedical Ethical Committee of Peking University (Approval No. LA ) Urine Sample Collection and Pretreatment Urine samples in each group (n = 6) were collected during the first 48 h after administration of the drugs began(blank urine collected by self-control.); Finally, all urine samples from the same group were merged into one sample, then dried under vacuum at 50 C using a Heidolph Laborota 4001 rotatory evaporator (Heidolph Instruments GmbH & Co., Schwabach, Germany), and then 1.00 g of the dried samples were reconstituted in 10 ml methanol, followed by 30 min ultrasonic extraction and 15 min centrifugation at 5,000 rpm. Afterward, the supernatant was collected for detection Instrumentations and Conditions HPLC analysis was performed on a Shimadzu HPLC (Shimadzu, Kyoto, Japan) equipped with two LC-20AD pumps, acto-20a column oven, an SIL-20AC autosampler, an SPD-M20A PDA detector and a CBM-20A system controller. The chromatographic separation was carried out on a Phenomenex Gemini C 18 column ( mm, 5 μm) protected with a Phenomenex Security Guard column (4 3.0 mm, 5 μm) (Phenomenex, Torrance, CA, USA). For each sample, an aliquot of 20 μl was injected with needle wash. The thermostatted auto-sampler was maintained at 15 C; column oven temperature was kept at 30 C. The column was eluted with a gradient mobile phase consisted of water-formic acid (100:0.1, v/v) (A) and acetonitrile (B) at the flow rate of ml/min. Gradient program was adopted in the following manner: 5% B at 0 10 min, 5% 15% B at min, 15% 40% B at min, 40% 65% B at min, 65% 100% B at min, 100% B at min, 5% B at min. High resolution mass spectra were recorded on an IT-TOF mass spectrometer (Shimadzu). The ESI source was operated both in negative and positive ion mode. The mass spectrometry was programmed to carry out full scan over m/z Da (MS 1 ), m/z Da (MS 2 and MS 3 ). A trifluoroacetic acid sodium solution (2.5 mm) was used to calibrate the mass range from 50 to 1000 Da. The other parameters were set as follows: flow rate, 0.20 ml/min (split from 1.00 ml/min HPLC effluent); heat block and curved desolvation line temperature, 200 C; nebulizing nitrogen gas flow, 1.5 L/min; interface voltage: (+), 4.5 kv; ( ), 3.5 kv; detector voltage, 1.70 kv; relative collision-induced dissociation energy (50%) [15].

21 Molecules 2014, Data Analysis All data were recorded and processed by Shimadzu software LCMS solution version 3.60, Formula Predictor version 1.2 and Accurate Mass Calculator (Shimadzu). 4. Conclusions A comparison was conducted on the similarities and differences of crude extracts and urine samples of DBT1 and DBT2. The chemical profiles of the crude extracts comprised a total of 115 proposed chemical components. There were 24 common ingredients, which was accounted for 27% in the total 91 identified components. There were a total of 56 isoflavonoids and saponins identified, which accounted for nearly 62% in the total identified components. Since isoflavonoid glycosides had acetylation (C18, C32, C34, C40, C44, C46, and C47), the formation of malonate acid esters (C31, C35, C43, C42, and C51) and special astragalosides (C49, C52, C53, C54, C60, and C64) in DBT1, the identified compounds from DBT1 were significantly greater than DBT2. Of these, C18, C34, C35, C42, C47, and C51 were identified from DBT for the first time. In total, 78 proposed chemical components in the urine samples of rats that had been administrated DBT1 and DBT2, respectively, were found. These included 19 common ingredients, which accounted for approximately 33% in the total identified constituents. In addition, 48 of total isoflavonoids and saponins were found, which accounted for nearly 82% of the total 59 identified components. The differences between those metabolites in the urine samples were revealed to be less than the crude extracts. These identified metabolites are mainly originated from formononetin, calycosin and their related glycosides, which are formed mainly through the metabolic processes of reduction, deglycosylation, demethylation, hydrogenation and sulfation. Through the comparison of chemical profiles of two DBTs at our used doses, the similarity of urine samples is higher than that of the crude extract samples, one can think the main chemical constituents are almost the same as when administrated to rats. The HPLC-DAD-ESI-IT-TOF-MS n method was successfully applied for the chemical profile comparison of two different DBTs and its related medicinal materials. The proposed assay provides an important reference and can be a suitable method for the rapid and accurate chemical basis evaluation of TCM or their related prescriptions. Supplementary Materials Supplementary materials can be accessed at: Acknowledgements This study was financially supported by Hong Kong Baptist University Faculty Research Foundation (Grant No. FRG2/12-13/058). We thank to Li Jun for HPLC-MS analysis in this work.

22 Molecules 2014, Authors Contributions Ya-Zhou Zhang, who presided the research work, finished the data analysis, and written this paper; Feng Xu, The main works by the writer are the data analysis, and participated in the writing of this paper; Tao Yi, who participated in the design of research and modified the paper; Jian-Ye Zhang, who participated the research design, and the data analysis; Jun Xu, who participated in the research design, and modified the paper; Yi-Na Tang, who participated in the research design and data analysis; Xi-Chen He, who participated in the data analysis of LC-MS; Jing Liu, who participated in the experiment work; Hu-Biao Chen, who is providing fund for this research work, the design of the work and modified the paper. Conflict of Interest The authors declare no conflict of interest. References 1. Zhang, W.L.; Choi, R.C.; Zhan, J.Y.; Chen, J.P.; Luk, W.K.; Yao, P.; Dong, T.T.; Tsim, K.W. Can Hedysari Radix replace Astragali Radix in Danggui Buxue Tang, a Chinese herbal decoction for woman aliment? Phytomedicine 2013, 20, Zheng, K.Y.Z.; Choi, R.C.Y.; Xie, H.Q.H.; Cheung, A.W.H.; Guo, A.J.Y.; Leung, K.W.; Chen, V.P.; Bi, C.W.C.; Zhu, K.Y.; Chan, G.K.L.; et al. The expression of erythropoietin triggered by Danggui Buxue Tang, a Chinese herbal decoction prepared from Radix Astragali and Radix Angelicae Sinensis, is mediated by the hypoxia-inducible factor in cultured HEK293T cells. J. Ethnopharmacol. 2010, 132, Yang, M.; Chan, G.C.F.; Deng, R.X.; Margaret, H.N.; Cheng, S.W.; Lau, C.P.; Ye J.Y.; Wang, L.J.; Liu, C. An herbal decoction of Radix astragali and Radix angelicae sinensis promotes hematopoiesis and thrombopoiesis. J. Ethnopharmacol. 2009, 124, Zhang, H.M.; Chen, S.W.; Deng, X.F.; Yang, X.G.; Huang, X. The effects of Danggui-Buxue- Tang on blood lipid and expression of genes related to foam cell formation in the early stage of atherosclerosis in diabetic GK rats. Diabetes Res. Clin. Pr. 2007, 77, Zhang, H.M.; Chen, S.W.; Deng, X.F.; Yang, X.G.; Huang, X. Danggui Buxue Tang decoction has an anti-inflammatory effect in diabetic atherosclerosis rat model. Diabetes Res. Clin. Pr. 2006, 74, Gao, J.; Huang, Y.; Li, P.; Xu, D.J.; Li, J.; Liu, Y.; Huang, Z.G.; Wu, Q.; Shao, X. Antifibrosis effects of total glucosides of Danggui-Buxue-Tang in a rat model of bleomycin-induced pulmonary fibrosis. J. Ethnopharmacol. 2011, 136, Xie, Q.F.; Xie, J.H.; Dong, T.T.X.; Su, J.Y.; Cai, D.K.; Chen, J.P.; Liu, L.F.; Li, Y.C.; Lai, X.P.; Tsim, K.W.K.; et al. Effect of a derived herbal recipe from an ancient Chinese formula, Danggui BuxueTang, on ovariectomized rats. J. Ethnopharmacol. 2012, 144, Gao, Q.T.; Choi, R.C.Y.; Cheung, A.W.H.; Zhu, J.T.T.; Li, J.; Chu, G.K.Y.; Duan, R.; Cheung, J.K.H.; Jiang, Z.Y.; Dong, X.B.; et al. Danggui Buxue Tang-A Chinese herbal decoction

23 Molecules 2014, activates the phosphorylations of extracellular signal-regulated kinase and estrogen receptor a in cultured MCF-7 cells. FEBS Lett. 2007, 581, Gao, Q.T.; Cheung, J.K.H.; Li, J.; Jiang, Z.Y.; Chu, G.K.Y.; Duan, R.; Cheung, A.W.H.; Zhao, K.J.; Choi, R.C.Y.; Dong, T.T.X.; et al. A Chinese herbal decoction, Danggui Buxue Tang, activates extracellular signal-regulated kinase in cultured T-lymphocytes. FEBS Lett. 2007, 581, Li, W.Z.; Li, J.; Bi, C.W.; Cheung, A.W.; Huang, W.; Duan, R.; Choi, R.C.; Chen, I.S.; Zhao, K.J.; Dong, T.T.; et al. Can rhizoma chuanxiong replace Radix angelia sinensis in the traditional chinese herbal decoction Danggui Buxue Tang? Planta Med. 2009, 75, Zhang, Y.Z.; Xu, F.; Liang, J.; Tang, J.S.; Shang, M.Y.; Wang, X.; Cai, S.Q. Isoflavonoids from the roots of Astragalus membranaceus var. Mongholicus. Zhongguo Zhong Yao Za Zhi 2012, 37, Fan, L.L.; Yi, T.; Xu, F.; Zhang, Y.Z.; Zhang, J.Y.; Li, D.P.; Xie, Y.J.; Qin, S.D.; Chen, H.B. Characterization of flavonoids in the ethomedicine fordiae cauliflorae radix and its adulterant millettiae pulchrae radix by HPLC-DAD-ESI-IT-TOF-MS n. Molecules 2013, 18, Li, C.Y.; Qi, L.W.; Li, P. Correlative analysis of metabolite profiling of Danggui Buxue Tang in rat biological fluids by rapid resolution LC-TOF/MS. J. Pharm. Biomed. Anal. 2011, 55, Yi, L.Z.; Liang, Y.Z.; Wu, H.; Yuan, D.L. The analysis of Radix Angelicae Sinensis (Danggui). J. Chromatogr. A 2009, 1216, Polat, E.; Bedir, E.; Perrone, A.; Piacente, S.; Alankus-Caliskan, O. Triterpenoid saponins from Astragalus wiedemannianus Fischer. Phytochemistry 2010, 71, Zhang, Y.Z.; Xu, F.; Dong, J.; Liang, J.; Hashi, Y.; Shang, M.Y.;Yang, D.H.; Wang, X.; Cai, S.Q. Profiling and identification of the metabolites of calycosin in rat hepatic 9000 g supernatant incubation system and the metabolites of calycosin-7-o-β-d-glucoside in rat urine by HPLC DAD ESI-IT-TOF-MS n technique. J. Pharm. Biomed. Anal. 2012, 70, Wen, X.D.; Liu, E.H.; Yang, J.; Li, C.Y.; Gao, W.; Qi, L.W.; Wang, C.Z.; Yuan, C.S.; Li, P. Identification of metabolites of Buyang Huanwu decoction in rat urine using liquid chromatography quadrupole time-of-flight mass spectrometry. J. Pharm. Biomed. Anal. 2012, Qi, L.W.; Cao, J.; Li, P.; Yu, Q.T.; Wen, X.D.; Wang, Y.X.; Li, C.Y.; Bao, K.D.; Ge, X.X.; Cheng, X.L. Qualitative and quantitative analysis of Radix Astragali products by fast high-performance liquid chromatography-diode array detection coupled with time-of-flight mass spectrometry through dynamic adjustment of fragmentor voltage. J. Chromatogr. A 2008, 1203, Liu, M.H.; Tong, X.; Wang, J.X.; Zou, W.; Cao, H.; Sua, W.W. Rapid separation and identification of multiple constituents in traditional Chinese medicine formula Shenqi Fuzheng Injection by ultra-fast liquid chromatography combined with quadrupole-time-of-flight mass spectrometry. J. Pharm. Biomed. Anal. 2013, 74, Liu, C.F.; Qiao, X.; Liu, K.D.; Miao, W.J.; Li, Y.J.; Liu, Y.; Jiang, Y.Y.; Bo, T.; Shi, R.B.; Guo, D.A.; et al. In vivo metabolites and plasma exposure of TongMai Keli analyzed by UHPLC/DAD/qTOF-MS and LC/MS/MS. J. Ethnopharmacol. 2013, 145,